; Bacillus anthracis is an endospore-forming, soil microorganism that is an important zoonotic pathogen. It is of significant concern as a potential bioterrorism agent. The spore is the infective form of this pathogen. The route by which the spore gains access to a human host defines the nature of the resulting disease, cutaneous, gastrointestinal, or pulmonary. Gastrointestinal and pulmonary anthrax are associated with high mortality rates. The B. anthracis spore possesses an outer layer referred to as the exosporium. This balloon-like outer layer contains the spore surface molecules that constitute the site of initial interactions with the host innate immune system. The exosporium is covered with a hair-like nap layer made up principally by the collagen-like glycoprotein BclA. This exosporium protein binds to receptors on phagocytic cells to promote uptake of the spores and which initiates the infectious process. Although it plays an important role in the initial stages of infection, very little is known regarding the composition, assembly process, and function of the exosporium layer. Much of what we know regarding the sporulation process was learned through biochemical and genetic studies of Bacillus subtilis, a model bacterium whose spores lack an exosporium. We propose to study exosporium composition in B. anthracis with a genetic and biochemical focused approach. A transposon mutagenesis system developed in my laboratory has produced a collection of potential exosporium, interspace, and outer spore coat mutants. These mutants will be characterized in this project. Additional outer spore proteins will be identified by a proteomic approach and by identification of proteins which form complexes with known exosporium proteins in sporulating cells. In addition to identifying exosporium genes and their products, identification of proteins present in larger complexes in sporulating cells will permit u to begin to determine which proteins interact in the sporulation process and we can begin to map the order of proteins in the exosporium assembly process. This project will lead to a better understanding of endospore maturation in B. anthracis and will identify spore targets that may prove useful for future vaccine or therapeutic development. In addition, a number of endospore-forming human pathogens possess exosporia, including Bacillus cereus, Clostridium perfringens, and Clostridium difficile and a more detailed understanding of the exosporium of B. anthracis may provide insights on spore biology for these other important human pathogens.